Spectroscopy Systems for Biodetection...College of Marine Science, University of South Florida Spectroscopy Systems for Biodetection Prof. L. H. Garcia-Rubio College of Marine Science,

College of Marine Science, University of South Florida

Spectroscopy Systems for Biodetection

Prof. L. H. Garcia-Rubio

College of Marine Science, Engineering, Physics, Chemistry, and Public Health,

University of South Florida, St. Petersburg, FL

The Future of Biodetection Systems, Santa Fe, NM, September 26-27, 2006

2College of Marine Science, University of South Florida

Research and Technology sponsors

• American Waterworks Research Foundation

• Avecor Cardiovascular• Baxter Biomedical• Beckman-Coulter

Corporation • Cargill Fertilizer • Constellation Technologies• Florida Blood Services• Florida Department of

Health• HF Scientific• ICI Paints• Instrumentation Metrics• ISP Chemicals• Kimberly-Clark

• Kodak• Los Alamos National Labs• Lucent Technologies• Negromex• NSF-ERC for Particle

Science and Technology University of Florida

• Ocean Optics Inc.• Ortho Clinical Diagnostics• Radiometer Medical A. C.• SC Johnson Polymers• USF College of Marine

Science• USF Center for Biological

Defense• Xerox Corporation


Graduate StudentsCătălina AlupoaeiAlicia García-López

Ph.D.

Daniel ImeokpariaSomnath ShettiJeff. ThrockmortonSylvia ChangSmita NarayananEsteban MarquezS. NarayananErik EstimleChristina BaconHaibin HuangYvette MattleyAndres Cardenas

Maria CelisSuresh ThennadilGita IranipoorMichelle JanowiakJudy FuVineet ShastryAkihisa NonoyamaYong-Rae KimEduardo Zurek

MSc.

Scott FisherStephanie RobertsonColleen O’Brian Lisa BruderPaul SacotoJaime Vara


Presentation overview

• Overview of Spectroscopy Methods• Chemical Markers• Physicochemical Markers• Recent Advances• Example: Uv-vis Spectroscopy • Vision for the Future


Applications of Spectrometer Systems for Biodetection

• Detection of Chemical Markers– Invasive (molecular)– Non-invasive (molecular-phenotype)

• Physicochemical Markers– Invasive (labelling)– Non-invasive (phenotype)

• Culture Methods (amplification)• Non-Culture Methods (direct)

• Laboratory Applications• Field (in-situ) Applications


Detection of Chemical Markers• GC-MS

– Carbohydrates – Polysaccharides, Lipolysacharides– Fatty Acids

• FT-IR– Total chemical and biochemical composition

• UV Resonance Raman Spectroscopy– Aminoacids, proteins, nucleic acids, pigments

• Multidimensional Fluorescence– Natural Fluorescence– Fluorescence Labelling

• Fluorescence Lifetimes• UV-Vis Spectroscopy


Detection of Chemical Markers

• Advantages:– High Sensitivity and Specificity to the identified

markers (Analytical Chemistry)– Trace Analysis Possible– Profiling of : carbohydrates, sugars, fatty acids,

etc.

• Issues:– Marker Identification– Sampling (cultures, environmental, clinical)– Sample preparation (derivatization, amplification)– Interferences: require fractionation GC, HPLC


Detection of Physicochemical Markers

• Angular Light Scattering– Static LS– Dynamic LS

• Microscopy– Fluorescence– FT-IR

• Imaging Methods• Hybrid Methods

– UV-Vis Spectroscopy


Detection of Physicochemical Markers

• Advantages:– High Sensitivity– Portable Instrumentation– Non-invasive

• Issues:– Marker Identification (size, shape, polarization)– Non-Specific markers– Sampling– Sample preparation– Interferences: require fractionation (Flow

Cytometry)


Analytical/Interpretation Methods

• Theoretical– Band assignment– Quantification

• Linear Models (Beer-Lambert)• Non-Linear Models (Mie theory)

• Statistical– Multivariate Statistical Models

• Factor Analysis• Principal Components• Genetic Algorithms

– Classification Algorithms• Pattern Recognition


Recent Advances

• Miniaturized Instrumentation– Detectors (MS, IR, Uv-vis, Raman)– Sampling and Sample preparation systems

• Microfluidics• Nano-technology

• Interpretation-Analysis– Improved Scattering Algorithms (T-Matrix)– Enhanced Computation Capabilities

• Improved Multivariate Statistical Models• Factor Analysis• Partial Least Squares• Pattern Recognition


BiophotonicsAnalyzer Requirements

• Portable laboratories– Autonomous– Miniaturized

• Sophisticated sensors & instrumentation• Multipurpose or multi-sensor capabilities• Immobilized reagent technologies• Rapid screening & diagnosis capabilities• Efficient communication subsystems• Local high performance processing capability


Implications of MiniaturizedFiber-Optics Spectrometry

• Measurements (spectrometer systems) can be taken to the process

• Sensors (fiber-optics) can be configured• Sensors (fiber-distal ends) can be designed for specific

applications (polymer & sol-gel immobilized reagents)• Detectors (spectrometers) can be configured• Design sampling-measurement systems with the objective of

increasing the information content of a measurement (detector and sensor bundles)


Reflectance/Backscattering

DetectorsSource

ProbeProbe

SourceProbe

Lens

Probe 2Probe 1

Source

Probe Source

Probe ConfigurationsProbe Configurations


% Saturation Wavelength (nm)

Ref

lect

ance

10 mm

5 mm

1 mm

Reflectance of Optically Dense Media for Three Penetration Depths


Fiber Optic Configuration

Fiber optic

Silane layerAcryloyl chloride

Hydrophilic copolymer with indicators

pH=6 pH=7

H+

H+

H+

pH=6 pH=5

H+

H+

H+


Spatial Patterns: the Belousov-Zhabotinsky Reaction


Endoscopy: application to fetal surgery

500 550 600 650 700 750 8000

2

4

6

8

10

12

14

16

18

Wavelength (nm)

Ref

lect

ance

, %

Arterial

Venous

500 550 600 650 700 750 8000

2

4

6

8

10

12

14

16

18

Wavelength (nm)

Ref

lect

ance

, %

Arterial

Venous

l l


Measurement PlatformSubsystemsDetector-Sensor ArrayElectronics PackCommunication UnitGlobal Positioning System

Prototype LMRA Platform



• Overview of Spectroscopy Methods• Chemical Markers• Physicochemical Markers• Recent Advances• Example: Uv-vis Spectroscopy• Vision for the Future


Particles Are Characterized by a Joint Property Distribution

Surface Charge

Joint Property Distribution

Size Distribution

Chemical Composition

Internal Structure

Shape Distribution


Issues in Bio-particle Characterization

Dilute Dispersions Enumeration Shape Composition Internal Structure Size Charge Orientation Mobility Optical properties Identification Limits of Detection

Concentrated DispersionsDense Media: skin, Suspensions, blood, cell cultures

Fluid structureParticle-Particle InteractionsHydrodynamic Interactions (colony forming)

SamplingSample IntegrityRepresentative Sampling

BackgroundGrowth Media Detection Limits (contrast)


Spectrometry of Complex Particles: Spectrometry of Complex Particles: PlateletsPlatelets

Dense Bodies:ADP/ATP (~1 M total)Calcium (2 M)Serotonin (0.065 M)Pyrophosphate (0.3 M)

Granule Diameter: 20 to 30 nm Platelet Diameter: 1.5 to 2.5 um



Advantages of Multiwavelength Spectroscopy

• large dynamic range (resolution: α/λ)• composition is accessible through

absorption• real time measurements (sample times <

0.01 sec)• large signal/noise• suitable for in situ, at-line and in-line

applications


Interpretation of spectral data

Scattering Models

ApproximationsRDG van de HulstApproximationsRDG van de Hulst

Mie TheoryMie Theory Other Methods: - T Matrix - DDA - MMP

Other Methods: - T Matrix - DDA - MMP

• Spherical & non-spherical particles • Form factors• non-absorbing particles

• Absorbing & non-absorbing particles• Spherical particles• Fractal Spheres • Computationally

intensive

Theoretical Methods & approximations for complex particles


Single Scattering Models (Mie)

Ωω−Ω

Ωπ

ω−=ωΩω−ΩΩΩ

π=−ω

λ=λλ

λπ=λε

λπ=α

λ+

λ=λ

απ=λτ

∫∫

∑∑

∫

∞∞

∞

d)(n2

)(k:d)(k2

1)(n

)j(p)j,(n)(n:)j(p)j,(k4

)(

D

n

)(ki

n

)(n)(m

dD)D(fD),m(Q4

N)(

022

022

M

j1obs1

M

j

1obs

o

1

o

1

0

2po l

l1

D

Macroscopicconcentratio

nNp, Qabs, &

Qsca

ParticleNp, Qabs, & Q sca

Par

ticle

Siz

e D

istr

ibut

ion

Optical P

roperties


Theoretical interpretation mTheoretical interpretation modelodel

6

Internal Structure Cytoplasm

Cell

=

Cytoplasm +Chromophores

Macrostructure

+

Chromophores

InternalStructure

∫∞

=0

200 )()),(()

4()( dDDfDDmQN extp λπλτ l


Typical Normalized Optical Density Spectra Typical Normalized Optical Density Spectra

Measured from Suspensions of Measured from Suspensions of MicroorganismsMicroorganisms

20


Comparison of Vegetative CellsComparison of Vegetative Cells

20


Measured Spectra: B. globigii

21

0

1

2

3

4

190 290 390 490 590 690 790Wavelength (nm)

No

rmal

ized

Op

tica

l Den

sity

Vegetative cellsSpores

-0.04

-0.02

0

230 270 310 350

Wavelength (nm)

Firs

t Der

ivat

ive


E. ColiE. Coli -- Difference of Optical DensityDifference of Optical Density

20


Growth behavior: Growth behavior: E. ColiE. Coli


Growth Behavior:Growth Behavior:PantoeaPantoeaagglomeransagglomerans((YersiniaYersinia PestisPestis))


Sporulation Media: B. Sporulation Media: B. SubtilisSubtilis


Deconvolution of the Cryptosporidium Deconvolution of the Cryptosporidium Transmission SpectrumTransmission Spectrum

0.0

1.0

2.0

3.0

4.0

100 200 300 400 500 600 700 800 900

Wavelength (nm)

Nor

mal

ized

Opt

ical

Den

sity

Measured

Calculated

Residuals

0.0

1.0

2.0

3.0

4.0

100 200 300 400 500 600 700 800 900

Wavelength (nm)

Nor

mal

ized

Opt

ical

Den

sity

Measured

Calculated

Cell

Internal Structure

0.0

1.0

2.0

3.0

4.0

100 200 300 400 500 600 700 800 900

Wavelength (nm)

Nor

mal

ized

Opt

ical

Den

sity

MeasuredCalculatedCellInternal StructureNucleic AcidProtein

0.0

1.0

2.0

3.0

4.0

100 200 300 400 500 600 700 800 900

Wavelength (nm)

Nor

mal

ized

Opt

ical

Den

sity

Measured


Quantitative Interpretation of the Quantitative Interpretation of the Transmission Spectrum for CryptosporidiumTransmission Spectrum for Cryptosporidium

MicroorganismAverage Size = 4.07 umParticle No = 341,000 per mL

Corpuscles/Organelles (internal structure)Hypochromicity = 0Average Size = 114 nmVolume Fraction = 0.4Fraction DNA+RNA+Nucleotides = 0.06Corpuscle Concentration = 5.21 ug/mL suspensionCorpuscle Enumeration = 6.46 billion/mL suspension

Max. No. of Corpuscles/cell = 46,481Estimated No corpuscles/cell = 18,950DNA+RNA+Nucleotides = 0.94 pg/cell


Chlamydia pneumoniaeinfected cells: HEp-2 (A); monocyte THP-1 (B, E); lymphoid Molt (C, F; lymphoid P3HR1 (D,G). Infected with 10 bacteria/cell after 72 Hrs of incubation. From Y. Yamamoto et al. private Comm. October 2002.


Representative data from the normalized optical density spectra of THP-1 Cells (human monocyte cell) infected

with C. pneumoniae

MOI = Chlamydia cells/Host cells, Efficiency of infection = 1% (Yamamoto)


Bacillus globigii (spores)

Quantitative Deconvolution

Volume, µm3

Literature values

0.782

<1

Equivalent diameter, µm 1.14

DNA+RNA, g/cell 1.52 x10-14

DPA, %

(% dry cell (Murrell, 1969))

6.22

(5.6-13.55)

Fraction of internal structure, % 41.47

Average size internal structure, nm 132


DNA+RNA Content (pg/cell)

Size (um)

1.851.9341.217

5.0115 x 64

HaploidDiploid

Quantitative DeconvolutionLiterature

Quantitative Deconvolution of the Spectral Fingerprint ofSaccharomyces Cerevisiae (Baker’s Yeast)


Schematic Representation for Classification StrategySchematic Representation for Classification Strategy

20

Absorption and Scattering Model

Data BaseStationary Phase Cultures

Elements of Classification

Define a Probabilityof Detection Model

Classification Strategy


PROPOSED MODELPROPOSED MODEL

6

Internal Cytoplasm + Structure Cytoplasm Chromophores Chromophores

+=

Cell Macrostructure Internal Structure

Organism Information (Size, Internal Structure, etc)


Data baseData base

MicroorganismsMicroorganisms::

1. Bacterium:

• Gram negative

• Gram positive

2. Intracellular Parasites

3. Yeast3. Yeast

4. Protozoan Parasites4. Protozoan Parasites

5. Viruses5. Viruses

6. Algae6. Algae

Measurement MatrixMeasurement Matrix•• WaterWater

•• BloodBlood

•• MensesMenses

•• PlateletsPlatelets

•• Wash waterWash water

Growth MediaGrowth Media•• Standard Growth MediaStandard Growth Media

•• BloodBlood

•• MensesMenses

•• Platelet rich PlasmaPlatelet rich Plasma

•• Sporulation MediaSporulation Media


Pathogens in data base

+ denotes strains with vegetative growth curves * denotes strains with spore formation over time 1. denotes strains grown in blood 2. denotes strains grown in platelets 3. denotes strains grown in menses

Genus Name Species Name Strains Comments Gram Negative Bacteria Escherichia +2 coli 10 +2 Pantoea + agglomerans 1 + Salmonella choleraesuis 7 Shigella sonnei 2 Shigella boydii 1 Shigella flexneri 1 Pseudomonas + aeruginosa 1 + Pseudomonas fluorescens 1 Gram Positive Bacteria Enterococus faecalis 1 Staphylococcus aureus 6 Staphylococcus hominis 1 Staphylococcus +2 epidermidis 1 +2 Listeria monocytogenes 3 Listeria seeligeri 1 Bacillus +* subtilis 1 +* Bacillus +* anthrasis sterne 1 +* Intercellular Parasites Chlamydia 1 pneumoniae 1 1 Yeast Candida 1,3 albicans 1 1,3 Protozoan Parasites Cryptosporidium parvum 1 Giardia muris 1 Giardia lamblia 1 Plasmodium1 vivax 1 1 Plasmodium1 falciparum 1 1 Viruses Enterovirus poliovirus 1 Group B Arbovirus dengue 1 Algae Synechecoccus sp. 1 Karenia brevis 1



• Overview of Spectroscopy Methods• Chemical Markers• Physicochemical Markers• Recent Advances• Example: Uv-vis Spectroscopy • Vision for the Future


Multi-Dimensional Spectroscopy

Length

W

++++++

------

Diameter

+++

+

+

+

+

++

Fluorescense

Polarization

Broad-band source Transmission


Features:• Flexibility in angular

measurements• Additional angles easily added• Backscattering measurements

(bifurcated fibers)• Polarized/unpolarized light• Superimposed fields easily applied• Suitable for on-line applications• Parallel and converging beam• Enhanced backscattering capability

Multidimensional spectrometer concept

light CCD #4

CCD #3

CCD #1

CCD #2

collimating lenses

available for

backscatter


MAMW Prototype LANL


UV-VIS MAMW RESPONSES: Particle Shape

Spherical Particles: D= 1.9 µm Peanut Shape Particles: 1.87 µm


UV-VIS MAMW RESPONSES: NORMAL & SICKLED RBC’s

Normal whole blood sample Sickled whole blood sample


Photograph of the Fluorescence Measurement set up for the Multidimensional Spectrometer


Miniaturized Spectrometers

Documents

Spectroscopy Systems for Biodetection...College of Marine Science, University of South Florida Spectroscopy Systems for Biodetection Prof. L. H. Garcia-Rubio College of Marine Science,